Normal epithelial GES-1 cells of human gastric mucosa (Cell Resource Center of Shanghai Academy of Sciences, Chinese Academy of Sciences, Shanghai, China) were cultured in wells or flasks at 37°C in an atmosphere containing 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; GE Healthcare Life Sciences, Little Chalfont, UK). The medium contained 10% (v/v) fetal bovine serum (FBS; GE Healthcare Life Sciences), 0.25 µg/ml amphotericin B, 0.1 mg/ml streptomycin and 100 U/ml penicillin.

The wild-type HP strain 26695 was obtained from the Chinese Center for Disease Control and Prevention (Beijing, China). HP was cultured for 72 h at 37°C in an atmosphere containing 5% CO₂ on commercial HP culture plates.

Following washing once with PBS, GES-1 cells were placed in 6-well plastic plates at a density of 3×10⁵ cells/well in 1 ml FBS-free DMEM. HP was restored by a swab from the agar plates and suspended in DMEM at an optical density of 0.6 at 600 nm, which corresponded to 3×10⁶ colony-forming U/ml. The HP was added to cells in DMEM without FBS and antibiotics at a multiplicity of infection (MOI) of 100:1, and co-cultured at 37°C and 5% CO₂ for 24 or 48 h. The morphology of HP-infected GES-1 cells was observed by Olympus CKX53 Inverted Fluorescence Microscope (magnification, ×40; Olympus Corporation, Tokyo, Japan).

Apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit (BD Biosciences; Becton, Dickinson and Company, Franklin Lakes, NJ, USA), according to the manufacturer’s protocols and analyzed by FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA). Specifically, following washing twice with PBS, cells were re-suspended in 100 µl 1X binding buffer (BD Biosciences; Becton, Dickinson and Company), and then incubated for 15 min with fluorescein isothiocyanate Annexin V and propidium iodide (each 5 µl; BD Biosciences; Becton, Dickinson and Company) at 37°C in the dark. Subsequently, 400 µl 1X binding buffer was added to each tube, followed by detection on a FACS Canto II flow cytometer (BD Biosciences; Becton, Dickinson and Company).

The primers of β-actin, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), p16, c-Myc, p53 and p21 designed by Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA, USA) and primers sequences were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Total RNA was isolated from HP-infected GES-1 cells using a TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and detected spectrophotometrically. The cDNA was prepared using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA). A mixture consisting of RNA (500 ng/µl; 2 µl), 5X M-MLV RT buffer (12 µl), dNTP mixes (2.5 mM; 6 µl), RNase inhibitor (30 U/µl; 1 µl), M-MLV RT (5 U/µl; 4 µl), Oligo dT(18) primer (500 ng/µl; 3 µl) and diethyl pyrocarbonate water (17 µl) was cultured at 37°C for 1 h. RT-qPCR was performed on an Eppendorf PCR system (Eppendorf, Hamburg, Germany). cDNA (1 µl), forward and reverse primers (each 0.5 µl), SYBR^® Premix ExTaq (8 µl; Takara Biotechnology Co., Ltd., Dalian, China) and ddH₂O (9 µl) were added to the mix, followed by 32 cycles of amplification as follows: Denaturation at 94°C for 2 min; 32 cycles of 94°C for 20 sec, 55°C for 35 sec, 72°C for 25 sec; and extension at 72°C for 5 min. The following primers were used: iNOS, forward, 5′-GAG-CTTCTACCTCAAGCTATC-3′, and reverse, 5′-CCTGATGTTGCCATTGTTGGT-3′; COX-2, forward, 5′-AGATCATCTCTGCCTGAGTATCTT-3′, and reverse, 5′-TTCAAATGAGATTGTGGGAAAATTGCT-3′; p21, forward, 5′-CCATGATGTTGATGCCCTAC-3′, and reverse, 5′-TTGCCTGCCTTCCTTTCT-3′; p53, forward, 5′-CAGTCTACCTCCCGCCATAA-3′, and reverse, 5′-CTCCCAAACATCCCTCACAG-3′; c-Myc, forward, 5′-GGGCTTTATCTAACTCGCTGTA-3′, and reverse, 5′-GCTATGGGCAAAGTTTCGTG-3′; p16, forward, 5′-GAAGAAAGAGGAGGGGCTGG-3′, and reverse, 5′-CTGCAGACCCTCTACCCACC-3′; and β-actin, forward, 5′- CC TG G CACCCAG CACA AT-3′, a nd reverse, 5′-GCTGATCCACATCTGCTGGAA-3′. Subsequently, fluorescence was detected. Specific amplification was guaranteed by dissociation curves. Each sample was tested in triplicate, and the mean Cq value was calculated (17).

The GES-1 cells from each group were collected, washed with PBS twice on ice and incubated with a Radioimmunoprecipitation Assay protein lysate (Beyotime Institute of Biotechnology, Beijing, China; cat. no. P0013B) containing 1% benzene sulfonyl fluoride for 30 min at 4°C. The protein concentration in the supernatant was then measured with a BCA protein concentration kit (Beyotime Institute of Biotechnology; cat. no. P0010). Proteins (30 µg) were treated by 10% SDS-PAGE electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were then immunoblotted with primary antibodies against β-actin (dilution, 1:2,000; cat. no. MAB8929), Cox-2 (dilution, 1:1,000; cat. no. AF4198), iNOS (dilution, 1:1,200; cat. no. MAB9502), p16 (dilution, 1:800; cat. no. AF5779), c-Myc (dilution, 1:1,000; cat. no. MAB3696), p53 (dilution, 1:1,600; cat. no. AF1355) and p21 (dilution, 1:1,000; cat. no. AF1047; R&D Systems, Inc., Minneapolis, MN, USA) overnight at 4°C, followed by cultivation with goat IgG horseradish peroxidase-conjugated antibody (R&D Systems, Inc.; dilution, 1:1,000; cat. no. HAF019) at 37°C for 60 min and Enhanced Chemiluminescence Western Blotting Detection reagents (cat. no. RPN2209; GE Healthcare Life Sciences). The bands were visualized using a LAS-4000 system (GE Healthcare Life Sciences) and were analyzed quantitatively using ImageJ v1.8.0 software (National Institutes of Health, Bethesda, MD, USA).

GES-1 cells cultured in 6-well plates were processed in lipopolysaccharide (LPS; 50 ng/ml; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China; cat. no. L8880) or HP (MOI of 100) medium for 24 or 48 h. The concentration of interleukin-8 (IL-8) and IL-23 in supernatants of stimulated cells was detected by IL-8 ELISA kit and IL-23 ELISA kits (Wuhan Boster Biological Technology, Ltd., Wuhan, China; cat. nos. EK0413 and EK1612, respectively), according to the manufacturer’s protocols. Cytokine concentrations were determined by a standard curve.

Following 24 or 48 h of culture at 37°C, the GES-1 cells were transferred using trypsinization, counted and seeded at 1×10⁵ cells/ml into 6-well plates, followed by overnight culture at 37°C to form confluent single layers. Wounds were then induced using a 100 µl pipette tip and imaged at 0, 24 or 48 h under Olympus CKX53 Inverted Fluorescence Microscope at magnification, ×100. The migration distance of the monolayer from the wounded area during this time period was detected and expressed as a migration index (the migrating distance following a stimulus relative to that of control cells). Each assay was conducted in triplicate and repeated at least five times.

Invasion assays were conducted in vitro, using Transwell plates (Costar; Corning, Inc., Corning, NY, USA) with 8-µm pores. After 2×10⁴ GES-1 cells were processed in LPS (50 ng/ml) or HP (MOI of 100) medium for 48 h, they were added to the upper chamber of Transwell plates with Matrigel (Costar; Corning, Inc.) and DMEM in the lower chamber of Transwell plate contained with 5% FBS. Following 24 or 48 h incubation, cells on the upper surface were removed using cotton wool and cells attached to the bottom were fixed with 75% methanol at 37°C for 15 min and stained with 0.5% crystal violet at 37°C for 30 min. Images were captured and the cells were counted using Olympus CKX53 Inverted Fluorescence Microscope at ×200 magnification.

GES-1 cultivated in 6-well plates were infected with HP for 24 or 48 h, and then harvested and rinsed twice with PBS containing 0.2% bovine serum albumin (cat. no. B7542; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Following being stained with phycoerythrin-labeled monoclonal cluster of differentiation 44 (CD44; dilution, 1:200; cat. no. 562818; BD Biosciences; Becton, Dickinson and Company) or allophycocyanin-labeled CD54 (dilution, 1:200; cat. no. 559771; BD Biosciences; Becton, Dickinson and Company) antibodies or the isotype controls (dilution, 1:200; cat. no. 560787; BD Biosciences; Becton, Dickinson and Company) at 37°C for 30 min, the cells were washed twice with PBS and fixed in 10% (v/v) formaldehyde-PBS at 37°C. Finally, cells were sorted and analyzed using a BD FACSCalibur with four-color fluorescence detection system (BD Biosciences; Becton, Dickinson and Company). Statistical data were analyzed by FlowJo 7.6 software. Mean fluorescence intensity and the percentage of positive cells were estimated after the values for the isotype controls were subtracted.

Tissues from 6 normal male patients, 6 male patients with HP⁺ gastritis and 6 male patients with HP⁺ gastric cancer (stomach adenocarcinoma) aged 32-65 years (mean ± SD, 44.3±8.9 years) were collected from Chongqing Cancer Institute (Chongqing, China) or Kunshan First People’s Hospital Affiliated to Jiangsu University (Suzhou, China) from January 2013 and January 2017. The HP infection status of normal patients, patients with gastritis and patients with gastric cancer was confirmed by a Carbon 13 breathing experiment because this experiment could indicate that individuals were suffering from HP infection. All protocols were approved by the Ethics Committee of the Chinese Hospital Association (Beijing, China) and written informed consent was provided by all participants in all experiments.

CD44 and CD54 in the membrane of gastric tissue cells from different participants were analyzed with an IHC assay. Briefly, all tissues were fixed with 4% formaldehyde (Beyotime Institute of Biotechnology; cat. no. P0099) at 37°C for 30 min, and then were embedded in paraffin and cut into slices with a histotome. Following xylene-deparaffinizing and rehydration through AR grade absolute ethanol to distilled water at 37°C, the 5-mm-thick microarray sections were stained with Hydrogen Peroxide Block (cat. no. ab64218; Abcam, Cambridge, UK). Following cultured with rabbit anti-CD44 (dilution, 1:100; cat. no. 550392; BD Biosciences; Becton, Dickinson and Company) and anti-CD54 antibodies (dilution, 1:100; cat. no. BBA17; R&D Systems, Inc.) at 37°C for 40 min, the sections were washed with PBS three times and cultivated with Biotin goat anti-rabbit IgG (dilution, 1:100; cat. no. 550338; BD Biosciences; Becton, Dickinson and Company) for 30 min at 37°C. Subsequently, following colorization with 3,3′-diaminobenzidine, the nuclei were counterstained lightly with hematoxylin at 37°C. Images were captured and the cells were counted using Olympus CKX53 Inverted Fluorescence microscope, at magnification, ×200. The immunostained sections were evaluated by two independent pathologists.

Results from three independent experiments are presented as the mean ± standard error of the mean (SEM). Data were processed on SPSS 16.0 for Windows (SPSS,Inc., Chicago, IL, USA) and tested using one-way analysis of variance (ANOVA). The one-way ANOVA and post-hoc Tukey’s test were conducted on GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) based on an assumption of normal distribution. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of HP on cell survival and physiology, the morphology and viability of HP-infected GES-1 cells was analyzed by microscopy and flow cytometry, respectively (Fig. 1). In vitro, the majority of GES-1 cells were in good condition, and only a small portion underwent HP infection-induced apoptosis, indicating that the virulence factor toxicity of HP could induce extensive apoptosis.

The host cells employ reactive oxygen species and nitric oxide to eliminate an invading pathogen, but this reaction mechanism could also regulate the inflammatory response of host cells (18). A total of 2 major enzymes (iNOS and COX-2) involved in the metabolism of HP-infected GES-1 cells (Fig. 2) were therefore examined using RT-qPCR and western blotting. The gray of bands was scanned on ImageJ, with β-actin as the standard. The gene and protein expression of iNOS and COX-2 were determined to be upregulated following infection.

Since immune cells are the main secretor of cytokines, the inflammatory response capacity of GES-1 cells following HP infection was detected by measuring inflammatory factors (IL-8 and IL-23) in the supernatant using ELISA (Fig. 3). IL-8 and IL-23 levels were significantly increased in infected cells, compared with the control or LPS-treated groups. These results indicated that normal gastric mucosal cells can also secrete a number of cytokines to inflammatorily respond to and resist the bacterial invasion.

Since long-term repeated HP infection could induce chronic gastritis, gastric ulcer or gastric cancer, the expression of cancer-associated genes and proteins, including p16, c-Myc, p53 and p21, was analyzed in HP-infected GES-1 cells using RT-qPCR and western blotting (Fig. 4). It was determined that the gene and protein expression levels of p16, c-Myc, p53 and p21 were all enhanced following HP infection, compared with the control or LPS-treated groups, indicating that HP may be a risk factor of gastric cancer.

Since HP infection could change the expression of cancer-associated genes, causing the host cells to become cancerous, the cell migration and invasion abilities of HP-infected GES-1 cells were investigated by wound-healing experiments (Fig. 5A and B) and Matrigel assay (Fig. 5C and D). As expected, the migration and invasion abilities of HP-infected GES-1 cells were significantly increased, compared with the control or LPS-treated groups.

CD44 and CD54 are associated with tumor invasion and metastasis, and are highly expressed in various malignant tumor types, including gastric cancer (19). The CD44/54 protein expression levels in HP-infected GES-1 cells were quantified by flow cytometry (Fig. 6). Results demonstrated that CD44 and CD54 were upregulated in HP-infected cells, and that CD54 protein levels were all overexpressed.

The expression of CD44 and CD54 proteins in the tissues of different participants was quantified by IHC (Fig. 7) and it was determined that they were increased in HP-infected tissues. The increased CD44 and CD54 were positively associated with the severity of the disease, indicating that HP infection may critically affect the expression of CD44/54.